Mungbean yellow mosaic disease is characterized by a bright yellow mosaic on the leaves of infected plants and causes significant losses to mungbean (Vigna radiata) crops. Yellow Mosaic Disease is a major threat for five important pulse crops Black gram, Mungbean, French bean, Pigeon pea and Soybean. It causes an annual loss of about $300 million. Soybean is one of the major pulse crops. It is highly prone to yellow mosaic disease and causes yield loss of 21-86% depending on severity. The causative agent of this disease is identified as Mungbean Yellow Mosaic India Virus (MYMIV), which is a member of Geminivirus group and Begomovirus family. The virus in India is transmitted through whitefly. Symptoms of the disease include small yellow diffused patches, which coalesce making the leaf completely yellow. Newer leaves show, yellow mosaic and mottling.
MYMIV, like most of the bipartite Geminivirus, contain two single stranded, covalently closed circular DNA as their genetic material. These are named as DNA-A and DNA-B, each of approximately 2.7 kb in size. DNA A encodes mainly 4 proteins, which play important role in replication and transcription of both the DNA components. DNA B encodes only two proteins, which help in the movement of the viral particles. They replicate through double stranded DNA intermediates in nuclei of mature plant cells.
To study many biological processes of these bipartite Begomoviruses, and to screen resistant variety in a population, plants need to be infected with the virus. For this purpose many infection methods have been developed such as (1) Virus transmission from an infected plant to uninfected plant with the help of whitefly (B. tabaci) (2) Mechanical inoculation of purified virus or viral DNA dimers from infected plant to healthy plant and (3) Inoculation of Agrobacterium suspension having partial or full dimers of DNA A and DNA B in Ti plasmid.
The whitefly mediated inoculation is the oldest method used for infecting plant. But some of the important bipartite Begomovirus are non transmissible to the other hosts by white flies and frequency of infection is low as it depends upon whitefly. Further, maintaining the stock of pure virus-containing whiteflies is very inconvenient and hazardous; and completely controlled introduction of insects to the plant is difficult. Screening of seed population is also not fully reliable in whitefly mediated infection; as the resistance may arise against whitefly itself (Grimsley, N., Hohn, B., Hohn, T., and Walden, R. (1986). “Agroinfection”, an alternative route for viral infection of plants by using the Ti plasmid. Proc. Natl. Acad. Sci. USA 83:3282-3286).
To overcome the difficulties of using insect vector for viral inoculations, mechanical methods were also tested in early studies. These methods involve inoculation of purified virus or dimers of DNA A and B to an uninfected plant via mechanical injuries (Etessami, P., Callis, R., Ellwood, S., Stanley, J., (1988). Delimitation of essential genes of cassava latent virus DNA 2. Nucleic Acid Res. 16, 4811-4829). In few plants such as Tomato 100% infection were obtained by using these methods. But results were not so promising. Other viruses like maize streak virus, beet curly top virus and squash leaf curl virus which are not transmitted mechanically an efficient method for infection is needed (Stanley, J., Markham, P. G., Callis, R. J., Pinner, M. S., (1986). The nucleotide sequence of an infectious clone of the geminivirus beet curly top virus. EMBO J. 5, 1761-1767).
Since most of the geminiviruses are not mechanically transmissible so new approaches such as Agrobacterium mediated inoculation was found to be reliable and suitable method for artificial infection of plants with these viruses (Grimsley, N., Hohn, B., Hohn, T., and Walden, R. (1986) “Agroinfection”, an alternative route for viral infection of plants by using the Ti plasmid. Proc. Natl. Acad. Sci. USA. 83:3282-3286). Using this method Petunia plants were inoculated with Tomato Golden Mosaic Virus (Rogers, S. G., Bisaro, D. M., Horsch, R. B., Fraley, R. T., Hoffmann, N. L., Brand, L., Elmer, J. S., and Lloyd, A. M. (1986) Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants. Cell 45:593-600). It was the first report of Agroinoculation of a Bipartite Begomovirus. This method uses the ability of Agrobacterium to transfer its Ti plasmid DNA from bacterial cell to plant chromosome by recombination (Wang, K; Herrera-Estrella, L; Van Montagu, M; Zambryski, P. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from agrobacterium to the plant genome. Cell. (1984) September; 38(2):455-462).
Generally for infecting the plants with the MYMIV, partial dimers or full dimers of DNA A and DNA B of a bipartite Begomovirus is cloned into T-DNA of two separate Ti plasmids and introduced into separate Agrobacterium culture. These two Agrobacterium cultures, having dimer of DNA A and dimer of DNA B are mixed in equal amount and used for plant inoculation called as co-Agroinoculation. After recombination and transfer of T-DNA to the plant chromosomes, unit-length Geminivirus genomes are released with the help of virus and plant proteins. This method is widely used for many Begomoviruses like Tomato Golden Mosaic Virus, Mungbean yellow mosaic virus Mungbean, Yellow Mosaic India Virus (Katoor S. Usharani, Baliji Surendranath, Qazi M. R. Haq and Varagur G. Malathi (2005), Infectivity analysis of a soybean isolate of Mungbean yellow mosaic India virus by agroinoculation. J Gen Plant Pathol 71:230-237) and Potato yellow mosaic virus and Cassava mosaic virus.
In a modified version of Agroinoculation method of bipartite Begomovirus, DNA A and DNA B partial tandem repeats were cloned on two separate Ti plasmids DNAs and introduced into same Agrobacterium cell and used for plant inoculation (Jacob S S, Vanitharani R, Karthikeyan A S, Chinchore Y, Thillaichidambaram P and Veluthambi K (2003) Mungbean yellow mosaic virus-Vi agroinfection by codelivery of DNA A and DNA B from one Agrobacterium strain; Plant Dis. 87 247-251).
Recent study of the infection of MYMIV on Soybean plant was carried out by inoculating the plant with mixed Agrobacterium cultures harboring separately partial dimers of DNA A and DNA B. The maximum efficiency of infection was found only 21% (Katoor S. Usharani, Baliji Surendranath, Qazi M. R. Haq and Varagur G. Malathi (2005) Infectivity analysis of a soybean isolate of Mungbean yellow mosaic India virus by agroinoculation. J. Gen. Plant Pathol. 71:230-237). Therefore there is need for a better system for infecting the plants with Mungbean Yellow Mosaic India Virus (MYMIV) that gives higher efficiency of infection of plants with the virus.